Our work focuses on three interrelated aspects of the mechanisms by which transforming growth factor beta (TGF-beta) triggers cessation of the cell cycle and induction of synthesis of extracellular matrix proteins, and the ways that these processes can be annulled in human malignancies. First, we recently identified novel types of mutations in both the types I and II TGF-beta receptors in human hematopoietic tumors, and we now want to find out how these mutations affect receptor structure and function. Specifically, we will determine the functional defects of the dominant negative D404G mutant type II TGF-beta receptor (Tbeta-RII) we recently identified in a cutaneous T cell lymphoma. Also, we showed recently that resistance to TGF-beta 1 in chronic lymphocytic leukemia (CLL) was associated with the presence of Tbeta-RI mRNA but loss of functional surface Tbeta-RI; expression of surface Tbeta-RII was normal. We will determine the mutation(s) in the Tbeta-RI or possibly in the Tbeta-RII genes in several isolates of TGF-beta- resistant CLL, and will determine the effects of these mutations on Tbeta-RI function. In the course of our studies on the biosynthesis, and oligomerization of Tbeta-RII and Tbeta-RI, we showed that Zn++ions bind to the cytosolic domain of Tbeta-RI and that Zn++ is essential for Tbeta-RI function. To explore this further, we will determine the number of Zn++ bound to the cytosolic domain of Tbeta-RI and the amino acids that ligate them, and will determine the role of bound Zn++ in the function of Tbeta-RI. Our major focus is using several robust expression cloning strategies we have developed to isolate novel genes encoding proteins that either activate or inhibit specific TGF-beta signal transduction pathways. In particular, we will clone proteins that affect only a subset of the processes normally induced by TGF-beta, such as expression of genes encoding extracellular matrix proteins or genes encoding cell cycle regulatory proteins. We may also clone by expression protein fragments that act in a dominant-negative fashion to block the function of the corresponding wild-type protein. Specifically, we will isolate and sequence cDNAs encoding proteins that (i) abolish the ability of TGF-beta to induce growth inhibition and/or apoptosis, (ii) that stimulate expression, in the absence of TGF-beta, of specific promoters, JunB, PAI, p15, and/or p21 - that are normally induced by TGF-beta in cells we are using, and (iii) that abolish the ability of TGF-beta to induce these promoters. Some of these strategies utilize green florescent protein and cell surface reporter genes that are activated by the indicated promoter. Also, we will clone by complementation the genes defective in three mutant lines of HT1080 cells in which TGF-beta is unable to induce the PAI promoter. Most importantly, we will isolate and sequence full-length clones of these novel proteins, and determine their function in TGF-beta signaling.